Water-soluble luminescent quantum dots and biomolecular conjugates thereof and related compositions and methods of use

ABSTRACT

The present invention provides a water-soluble luminescent quantum dot, a biomolecular conjugate thereof and a composition comprising such a quantum dot or conjugate. Additionally, the present invention provides a method of obtaining a luminescent quantum dot, a method of making a biomolecular conjugate thereof, and methods of using a biomolecular conjugate for ultrasensitive nonisotopic detection in vitro and in vivo.

[0001] This application claims priority to U.S. provisional patentapplication Serial No. 60/101,748, filed Sep. 24, 1998, and U.S.provisional patent application Serial No. 60/131,987, filed Apr. 30,1999.

GOVERNMENT SUPPORT

[0002] This invention was made, in part, with funding from the NationalScience Foundation under Grant No. CHE-b 9610254 and from the Departmentof Energy under Grant No. FG02-98ER 24873. Therefore, the United Statesof America may have certain rights in the invention.

TECHNICAL FIELD OF INVENTION

[0003] The present invention relates to a water-soluble luminescentquantum dot, a biomolecular conjugate thereof and a compositioncomprising such a quantum dot or conjugate. Additionally, the presentinvention relates to a method of obtaining a luminescent quantum dot, amethod of making a biomolecular conjugate thereof, and methods of usinga biomolecular conjugate for ultrasensitive nonisotopic detection invitro and in vivo.

BACKGROUND OF THE INVENTION

[0004] The development of sensitive nonisotopic detection systems foruse in biological assays has significantly impacted many research anddiagnostic areas, such as DNA sequencing, clinical diagnostic assays,and fundamental cellular and molecular biology protocols. Currentnonisotopic detection methods are mainly based on organic reportermolecules that undergo enzyme-linked color changes or are fluorescent,luminescent, or electroactive (Kricka; Ed., Nonisotopic Probing,Blotting, and Sequencing, Academic Press, New York, 1995; Issac, Ed.,Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana,Totowa, N.J., 1994; and Diamandis and Christopoulos, Eds., Immunoassay,Academic Press, New York, 1996). While these nonisotopic systems solvethe problems associated with radioisotopic detection, such as shorthalf-lives of radioisotopes, health hazards and expensive removal ofradioactive waste, they are not as sensitive or stable as nonisotopicdetection systems that utilize luminescent semiconductor quantum dots.For example, highly luminescent semiconductor quantum dots, such asZnS-capped CdSe quantum dots, are twenty times brighter, one hundredtimes more stable against photobleaching, and three times narrower inspectral line width than organic dyes, such as fluorescent rhodamine.

[0005] Over the past decade, much progress has been made in thesynthesis and characterization of a wide variety of semiconductorquantum dots. Recent advances have led to large-scale preparation ofrelatively monodisperse quantum dots (Murray et al., J Am. Chem. Soc.,115, 8706-15 (1993); Bowen Katari et al., J Phys. Chem., 98, 4109-17(1994); and Hines et al., J Phys. Chem., 100, 468-71 (1996)). Otheradvances have led to the characterization of quantum dot latticestructures (Henglein, Chem. Rev., 89, 1861-73 (1989); and Weller et al.,Chem. Int. Ed. Engl. 32, 41-53(1993)) and also to the fabrication ofquantum-dot arrays (Murray et al., Science, 270, 1335-38 (1995); Andreset al., Science, 273, 1690-93 (1996); Heath et al., J Phys. Chem., 100,3144-49 (1996); Collier et al., Science, 277, 1978-81 (1997); Mirkin etal., Nature, 382, 607-09 (1996); and Alivisatos et al., Nature, 382,609-11 (1996)) and light-emitting diodes (Colvin et al., Nature, 370,354-57 (1994); and Dabbousi et al., Appl. Phys. Let., 66, 1316-18(1995)). In particular, IIB-VIB semiconductors have been the focus ofmuch attention, leading to the development of a CdSe quantum dot thathas an unprecedented degree of monodispersivity and crystalline order(Murray (1993), supra).

[0006] Further advances in luminescent quantum dot technology haveresulted in a dramatic enhancement of the fluorescence efficiency andstability of the quantum dots. The remarkable luminescent properties ofquantum dots arise from quantumsize confinement, which occurs when metaland semiconductor core particles are smaller than their exciton Bohrradii, about 1 to 5 nm (Alivisatos, Science, 271, 933-37 (1996);Alivisatos, J Phys. Chem., 100, 13226-39 (1996); Brus, Appl Phys., A 53,465-74 (1991); Wilson et al., Science, 262, 1242-46 (1993); Henglein(1989), supra; and Weller (1993), supra). Recent work has shown thatimproved luminescence can be achieved by capping a size-tunable lowerband gap core particle with a higher band gap shell. For example, CdSequantum dots passivated with a ZnS layer are strongly luminescent (35 to50% quantum yield) at room temperature, and their emission wavelengthcan be tuned from blue to red by changing the particle size. Moreover,the ZnS capping protects the core surface and leads to greater stabilityof the quantum dot (Hines (1996), supra; and Dabbousi et al., J Phys.Chem. B 101, 9463-75 (1997)).

[0007] Despite the remarkable advances in luminescent quantum dottechnology, the capped luminescent quantum dots are not suitable forbiological applications because they are not water-soluble. In addition,it has not been possible to attach a quantum dot to a biomolecule insuch a manner as to preserve the biological activity of the biomolecule.However, because luminescent quantum dots offer significant advantagesover currently available nonisotopic detection systems, there remains anunfulfilled desire for a luminescent quantum dot that can be used fordetection purposes in biological assays. In view of this, it is anobject of the present invention to provide a luminescent quantum dotthat is suitable for biological applications. It is another object ofthe present invention to provide a biomolecular conjugate of aluminescent quantum dot that is suitable for biological applications. Inparticular, the present invention seeks to provide a biomolecularconjugate of a luminescent quantum dot in which the biomolecule retainsits biological activity and the resultant conjugate is suitable forbiological applications. Accordingly, it is yet another object of thepresent invention to provide a method of making such a luminescentquantum dot and a method of making a biomolecular conjugate thereof.Still yet another object of the present invention is to provide acomposition comprising such a quantum dot or a biomolecular conjugatethereof. A further object of the present invention is to provide methodsof using the biomolecular conjugate for ultrasensitive nonisotopicdetection in vitro and in vivo. These and other objects and advantages,as well as additional inventive features, of the present invention willbecome apparent to one of ordinary skill in the art upon reading thedetailed description provided herein.

BRIEF SUMMARY OF THE PRESENT INVENTION

[0008] The present invention provides a water-soluble luminescentquantum dot, which comprises a core, a cap and a hydrophilic attachmentgroup. Also provided is a composition comprising the water-solubleluminescent quantum dot and an aqueous carrier. The present inventionfurther provides a conjugate, which comprises the water-solubleluminescent quantum dot and a biomolecule, wherein the biomolecule isattached directly or indirectly to the hydrophilic attachment group.Also provided is a composition comprising the conjugate and an aqueouscarrier.

[0009] Further provided by the present invention are a method ofobtaining a watersoluble luminescent quantum dot and methods of makingbiomolecular conjugates thereof. Other methods provided by the presentinvention include methods of detecting biomolecules in vitro and invivo.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1 is a schematic diagram of a bioconjugate comprising asingle-stranded oligonucleotide having a stem and loop structure and thebioconjugate bound to a target nucleic acid.

[0011]FIG. 2 is a schematic diagram of a method of detecting multiplenucleic acids in a sample in accordance with the present invention.

[0012]FIG. 3 is a schematic diagram of a method of detecting a viralnucleic acid in a sample in accordance with the present invention.

[0013]FIG. 4 is a schematic diagram of bioconjugates comprising aluminescent semiconductor quantum dot and an oligonucleotide and thebioconjugates bound by a DNA linker.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0014] The present invention provides means and methods ofultrasensitive nonisotopic detection of biomolecules in vitro and invivo. The present invention is premised on the surprising, unexpectedand advantageous discovery that capped luminescent quantum dots can berendered water-soluble by attaching a hydrophilic attachment group tothe cap of the quantum dot and that these quantum dots retain theirluminescent properties. The present invention is also premised on thediscovery that various biomolecules can be directly and indirectlyattached to the hydrophilic attachment group on the cap of the quantumdot and that these biomolecules can retain their biological activity.

[0015] In view of the above, the present invention provides, in oneembodiment, a water-soluble luminescent semiconductor quantum dot, whichcomprises a core, a cap and a hydrophilic attachment group. The “core”is a nanoparticle-sized semiconductor. While any core of the IIB-VIB,IIIB-VB or IVB-IVB semiconductors can be used in the context of thepresent invention, the core must be such that, upon combination with acap, a luminescent quantum dot results. A IIB-VIB semiconductor is acompound that contains at least one element from Group IEB and at leastone element from Group VIB of the periodic table, and so on. Preferably,the core is a IIB-VIB, IIIB-VB or IVB-IVB semiconductor that ranges insize from about 1 nm to about 10 nm. The core is more preferably aIIB-VIB semiconductor and ranges in size from about 2 nm to about 5 nm.Most preferably, the core is CdS or CdSe. In this regard, CdSe isespecially preferred as the core, in particular at a size of about 4.2nm.

[0016] The “cap” is a semiconductor that differs from the semiconductorof the core and binds to the core, thereby forming a surface layer onthe core. The cap must be such that, upon combination with a givensemiconductor core, results in a luminescent quantum dot. The cap shouldpassivate the core by having a higher band gap than the core. In thisregard, the cap is preferably a IIB-VIB semiconductor of high band gap.More preferably, the cap is ZnS or CdS. Most preferably, the cap is ZnS.In particular, the cap is preferably ZnS when the core is CdSe or CdSand the cap is preferably CdS when the core is CdSe.

[0017] The “attachment group” as that term is used herein refers to anyorganic group that can be attached, such as by any stable physical orchemical association, to the surface of the cap of the luminescentsemiconductor quantum dot and can render the quantum dot water-solublewithout rendering the quantum dot no longer luminescent. Accordingly,the attachment group comprises a hydrophilic moiety. Preferably, theattachment group enables the hydrophilic quantum dot to remain insolution for at least about one hour. More preferably the attachmentgroup enables the hydrophilic quantum dot to remain in solution for atleast about one day. Even more preferably, the attachment group allowsthe hydrophilic quantum dot to remain in solution for at least about oneweek, most preferably for at least about one month. Desirably, theattachment group is attached to the cap by covalent bonding and isattached to the cap in such a manner that the hydrophilic moiety isexposed. Preferably, the hydrophilic attachment group is attached to thequantum dot via a sulfur atom. More preferably, the hydrophilicattachment group is an organic group comprising a sulfur atom and atleast one hydrophilic attachment group. Suitable hydrophilic attachmentgroups include, for example, a carboxylic acid or salt thereof, asulfonic acid or salt thereof, a sulfamic acid or salt thereof, an aminosubstituent, a quaternary ammonium salt, and a hydroxy. The organicgroup of the hydrophilic attachment group of the present invention ispreferably a C₁-C₆ alkyl group or an aryl group, more preferably a C₁-C₆alkyl group, even more prefeably a C₁-C₃ alkyl group. Therefore, in apreferred embodiment, the attachment group of the present invention is athiol carboxylic acid or thiol alcohol. More preferably, the attachmentgroup is a thiol carboxylic acid. Most preferably, the attachment groupis mercaptoacetic acid.

[0018] Accordingly, a preferred embodiment of a water-solubleluminescent semiconductor quantum dot is one that comprises a CdSe coreof about 4.2 nm in size, a ZnS cap and an attachment group. Anotherpreferred embodiment of a watersoluble luminescent semiconductor quantumdot is one that comprises a CdSe core, a ZnS cap and the attachmentgroup mercaptoacetic acid. An especially preferred water-solubleluminescent semiconductor quantum dot comprises a CdSe core of about 4.2nm, a ZnS cap of about 1 nm and a mercaptoacetic acid attachment group.

[0019] In another embodiment, the present invention also provides acomposition comprising a water-soluble luminescent semiconductor quantumdot as described above and an aqueous carrier. Any suitable aqueouscarrier can be used in the composition. Desirably, the carrier rendersthe composition stable at a desired temperature, such as roomtemperature, and is of an approximately neutral pH. Examples of suitableaqueous carriers are known to those of ordinary skill in the art andinclude saline solution and phosphate-buffered saline solution (PBS).

[0020] In yet another embodiment, the present invention provides aconjugate comprising a water-soluble luminescent semiconductor quantumdot as described above and a biomolecule, wherein the biomolecule isattached to the quantum dot via the hydrophilic attachment group. Thebiomolecule should not render the quantum dot water-insoluble.Preferably, the biomolecule is a protein, a fragment of a protein, or anucleic acid. Use of the phrase “protein or a fragment thereof” isintended to encompass a protein, a glycoprotein, a polypeptide, apeptide, and the like, whether isolated from nature, of viral,bacterial, plant or animal (e.g., mamnmalian, such as human) origin, orsynthetic. A preferred protein or fragment thereof for use as abiomolecule in the present inventive conjugate is an antigen, an epitopeof an antigen, an antibody, or an antigenically reactive fragment of anantibody. Use of the phrase “nucleic acid” is intended to encompass DNAand RNA, whether isolated from nature, of viral, bacterial, plant oranimal (e.g., mammalian, such as human) origin, synthetic,single-stranded, double-stranded, comprising naturally or nonnaturallyoccurring nucleotides, or chemically modified. A preferred nucleic acidis a single-stranded oligonucleotide comprising a stem and loopstructure and the hydrophilic attachment group is attached to one end ofthe single-stranded oligonucleotide and a quenching moiety is attachedto the other end of the single-stranded oligonucleotide and thequenching moiety quenches the luminescent semiconductor quantum dot.

[0021] The biomolecule can be attached, such as by any stable physicalor chemical association, to the hydrophilic attachment group of thewater-soluble luminescent quantum dot directly or indirectly by anysuitable means. Desirably, the biomolecule is attached to the attachmentgroup directly or indirectly through one or more covalent bonds. If thebiomolecule is attached to the hydrophilic attachment group indirectly,the attachment preferably is by means of a “linker.” Use of the term“linker” is intended to encompass any suitable means that can be used tolink the biomolecule to the attachment group of the water-solubleluminescent quantum dot. The linker should not render the water-solubleluminescent quantum dot water-insoluble and should not adversely affectthe luminescence of the quantum dot. Also, the linker should notadversely affect the function of the attached biomolecule. If theconjugate is to be used in vivo, desirably the linker is biologicallycompatible.

[0022] For example, if the attachment group is mercaptoacetic acid and anucleic acid biomolecule is being attached to the attachment group, thelinker preferably is a primary amine, a thiol, streptavidin,neutravidin, biotin, or a like molecule. If the attachment group ismercaptoacetic acid and a protein biomolecule or a fragment thereof isbeing attached to the attachment group, the linker preferably isstrepavidin, neutravidin, biotin, or a like molecule. In accordance withthe invention, the linker should not contact the protein biomolecule ora fragment thereof at an amino acid which is essential to the functionor activity of the attached protein. Crosslinkers, such as intermediatecrosslinkers, can be used to attach a biomolecule to the attachmentgroup of the water-soluble luminescent quantum dot.Ethyl-3-(dimethylaminopropyl) carbodiimide (EDAC) is an example of anintermediate crosslinker. Other examples of intermediate crosslinkersfor use in the present invention are known in the art. See, for example,Bioconjugate Techniques (Academic Press, New York, (1996)).

[0023] Catalytic crosslinkers also can be used to attach a biomoleculeto the attachment group of the water-soluble luminescent quantum dot.Catalytic crosslinkers effect direct attachment of the biomolecule tothe attachment group. Examples of catalytic crosslinkers are also knownin the art. See, for example, Bioconjugate Techniques (1996), supra.

[0024] Attachment of a biomolecule to the attachment group of thewater-soluble luminescent quantum dot also can be effected by abi-functional compound as is known in the art. See, for example,Bioconjugate Techniques (1996), supra.

[0025] In those instances where a short linker could cause sterichindrance problems or otherwise affect the functioning of thebiomolecule, the length of the linker can be increased, e.g., by theaddition of from about a 10 to about a 20 atom spacer, using procedureswell-known in the art (see, for example, Bioconjugate Techniques (1996),supra). One possible linker is activated polyethylene glycol, which ishydrophilic and is widely used in preparing labeled oligonucleotides.

[0026] Accordingly, a preferred conjugate in accordance with the presentinvention is a conjugate comprising a CdSe core of about 4.2 nm, a ZnScap, a hydrophilic attachment group and a biomolecule. Another preferredconjugate in accordance with the present invention is a conjugatecomprising a CdSe core, a ZnS cap, a mercaptoacetic acid attachmentgroup and a biomolecule. An especially preferred conjugate comprises aCdSe core of about 4.2 nm, a ZnS coating of about 1 nm, a mercaptoaceticacid attachment group and a biomolecule.

[0027] An alternatively preferred conjugate in accordance with thepresent invention is a conjugate essentially as described above, whereinthe biomolecule is a single-stranded oligonucleotide comprising a stemand a loop. The hydrophilic attachment group is attached to one end ofthe single-stranded oligonucleotide, and a quenching moiety is attachedto the other end of the single-stranded oligonucleotide. The quenchingmoiety quenches the luminescent semiconductor quantum dot.

[0028] Any suitable quenching moiety that quenches the luminescence ofthe quantum dot can be used in the alternatively preferred conjugatedescribed above. Preferably, the alternatively preferred conjugatecomprises a primary amine group at the b 3′ end and a biotin group atthe 5′ end. The quenching moiety is preferably a nonfluorescent organicchromophore, which is covalently linked to the 3′ amino group of theoligonucleotide. More preferably, the quenching moiety is4-[4′dimethylaminophenylazo]benzoic acid (DABCYL). Preferably, theluminescent quantum dot of the bioconjugate is first derivatized withstreptavidin according to well-known cross-linking methods and thenconjugated to the 5′ biotin group, preferably at a 1:1 molar ratio.

[0029] Thus, in another embodiment, the present invention also providesa composition comprising a conjugate as described above and an aqueouscarrier. Any suitable aqueous carrier can be used in the composition.Desirably, the carrier renders the composition stable at a desiredtemperature, such as room temperature, and is of an approximatelyneutral pH. Examples of suitable aqueous carriers are known to those ofordinary skill in the art and include saline solution and PBS.

[0030] In view of the above, the present invention further provides amethod of obtaining a water-soluble luminescent semiconductor quantumdot as described. The method comprises (a) reacting a luminescentsemiconductor quantum dot as described above in a nonpolar organicsolvent with a first aqueous solution comprising an attachment group;(b) adding a second aqueous solution of about neutral pH and mixing; and(c) extracting an aqueous layer, thereby obtaining a water-solubleluminescent semiconductor quantum dot. Preferably, the nonpolar organicsolvent is chloroform and the attachment group is mercaptoacetic acid.

[0031] The present invention also provides a method of making aconjugate comprising a water-soluble luminescent semiconductor quantumdot and a biomolecule as described above. Where the biomolecule is to bedirectly attached to the attachment group of the quantum dot, the methodcomprises (a) contacting a water-soluble luminescent semiconductorquantum dot as described above with a biomolecule, which can directlyattach to the attachment group on the cap of the water-solubleluminescent semiconductor quantum dot; and (b) isolating the conjugate.Preferably, the biomolecule is a protein or a fragment thereof or anucleic acid. In one embodiment of the method of directly attaching thebiomolecule to the attachment group, the attachment group ismercaptoacetic acid and the biomolecule is a protein. In anotherembodiment of the direct attachment method, the quantum dot and thebiomolecule are contacted in the presence of a catalytic crosslinker.

[0032] Where the biomolecule is to be indirectly attached to theattachment group of the water-soluble luminescent semiconductor quantumdot, the present invention provides a method comprising (a) contacting awater-soluble semiconductor luminescent quantum dot as described abovewith a linker, which can attach to the attachment group and thebiomolecule; (b) isolating the water-soluble luminescent semiconductorquantum dot to which is attached a linker; (c) contacting thewatersoluble luminescent semiconductor quantum dot to which is attacheda linker with a biomolecule; and (d) isolating the conjugate.

[0033] Alternatively, the method comprises (a) contacting a biomoleculewith a linker, which can attach to the attachment group and thebiomolecule; (b) isolating the biomolecule to which is attached alinker; (c) contacting the biomolecule to which is attached a linkerwith a water-soluble luminescent quantum dot; and (d) isolating theconjugate. In one embodiment of the method of indirectly attaching thebiomolecule to the attachment group, the linker is a primary amine orstreptavidin, the attachment group is mercaptoacetic acid and thebiomolecule is a nucleic acid.

[0034] In another embodiment of the method of indirectly attaching thebiomolecule to the attachment group, the method comprises (a) contactinga water-soluble luminescent quantum dot with an intermediate crosslinkeror a bifunctional molecule, either one of which can attach to theattachment group and the biomolecule; (b) isolating the water-solubleluminescent quantum dot to which is attached the intermediatecrosslinker or the bifunctional molecule; (c) contacting thewater-soluble luminescent quantum dot to which is attached theintermediate crosslinker or the bifunctional molecule with abiomolecule; and (d) isolating the conjugate.

[0035] Alternatively, the method comprises (a) contacting a biomoleculewith an intermediate crosslinker or a bifunctional molecule, either oneof which can attach to the attachment group and the biomolecule; (b)isolating the biomolecule to which is attached the intermediatecrosslinker or the bifunctional molecule; (c) contacting the biomoleculeto which is attached the intermediate crosslinker or the bifunctionalmolecule with a water-soluble luminescent quantum dot; and (d) isolatingthe conjugate. An example of such an embodiment is a method employingmercaptoacetic acid as the attachment group, a protein or a fragmentthereof as the biomolecule, and EDAC as the intermediate crosslinker.

[0036] Also provided by the present invention is a method of detecting aprotein in a sample. The method comprises (a) contacting the sample witha conjugate as described above, wherein the biomolecule of the conjugatespecifically binds to the protein; and (b) detecting luminescence,wherein the detection of luminescence indicates that the conjugate boundto the protein in the sample.

[0037] Preferably, in the method of protein detection, the biomoleculeof the conjugate is a protein or a fragment thereof, such as an antibodyor an antigenically reactive fragment thereof, and the protein in thesample is an antigen or an epitope thereof that is bound by the antibodyor an antigenically reactive fragment thereof. The antigen or epitopethereof preferably is part of a virus or a bacterium. Alternatively andpreferably, the biomolecule of the conjugate is an antigen or an epitopethereof and the protein in the sample is an antibody or an antigenicallyreactive fragment thereof that binds to the antigen or epitope thereof.The antibody or the antigenically reactive fragment thereof preferablyis specific for a virus, a bacterium, or a part of a virus or abacterium. In yet another alternative and preferred embodiment, thebiomolecule of the conjugate is a nucleic acid and the protein in thesample is a nucleic acid binding protein, e.g., a DNA binding protein.

[0038] Another method provided by the present invention is a method ofdetecting a nucleic acid in a sample. The method comprises (a)contacting the sample with a conjugate as described above, wherein thebiomolecule of the conjugate specifically binds to the nucleic acid; and(b) detecting luminescence, wherein the detection of luminescenceindicates that the conjugate bound to the nucleic acid in the sample.Preferably, the biomolecule of the conjugate is a nucleic acid.Alternatively and preferably, the biomolecule of the conjugate is aprotein or a fragment thereof that binds to a nucleic acid, such as aDNA binding protein.

[0039] As shown in FIG. 1, the present invention also provides anothermethod of detecting a nucleic acid in a sample. This method involves theuse of a bioconjugate comprising a single-stranded oligonucleotidehaving a stem-and-loop structure, a quantum dot moiety, and a quenchingmoiety. The loop of the oligonucleotide comprises a probe sequence thatis complementary to a target sequence in the nucleic acid to be detectedin a sample. Desirably, the loop is of sufficient size such that itopens readily upon contact with a target sequence, yet not so large thatit is easily sheared. Preferably, the loop is from about 10 nucleotidesto about 30 nucleotides and more preferably from about 15 nucleotides toabout 25 nucleotides. The probe sequence can comprise all or less thanall of the loop. Preferably, the probe sequence is at least about 15nucleotides in length. The stem is formed by the annealing ofcomplementary sequences that are at or near the two ends of thesingle-stranded oligonucleotide. A luminescent quantum dot moiety iscovalently linked to one end of the single-stranded oligonucleotide anda quenching moiety is covalently linked to the other end of thesingle-stranded oligonucleotide. The stem keeps the luminescent quantumdot and quenching moieties in close proximity to each other so that theluminescence of the quantum dot is quenched when the single-strandedoligonucleotide is not bound to a target sequence. In this regard, thecomplementary sequences of which the stem is comprised must besufficiently close to the ends of the oligonucleotide as to effectquenching of the luminescent quantum dot. When the probe sequenceencounters a target sequence in a nucleic acid to be detected in asample, it binds, i.e., hybridizes, to the target sequence, therebyforming a probe-target hybrid that is longer and more stable than thestem hybrid. The length and rigidity of the probe-target hybrid preventsthe simultaneous formation of the stem hybrid. As a result, thestructure undergoes a spontaneous conformational change that forces thestem to open, thereby separating the quantum dot moiety and thequenching moiety and restoring luminescence.

[0040] Accordingly, the method comprises (a) contacting the sample witha conjugate, in which the biomolecule is a single-strandedoligonucleotide comprising a stem-and-loop structure and in which thehydrophilic attachment group is attached to one end of thesingle-stranded oligonucleotide and a quenching moiety is attached tothe other end of the single-stranded oligonucleotide, such that thequenching moiety quenches the luminescent semiconductor quantum dot, allas described above. The loop comprises a probe sequence that binds to atarget sequence in the nucleic acid in the sample. Upon binding, theconjugate undergoes a conformational change that forces the stem toopen, thereby separating the quantum dot and the quenching moiety. Themethod further comprises (b) detecting luminescence. The detection ofluminescence indicates that the conjugate bound to the nucleic acid inthe sample.

[0041] As shown in FIG. 2, the present invention provides yet anothermethod of detecting a single-stranded nucleic acid, such as mRNA, cDNA,or denatured doublestranded DNA in a sample, by attachment to a solidsupport, such as a membrane, glass bead, transparent polymer and thelike. The method comprises (a) contacting a sample comprising a firstsingle-stranded nucleic acid with a solid support to which is attached asecond single-stranded nucleic acid that can bind to the firstsingle-stranded nucleic acid, (b) contacting the solid support with aconjugate as described above, in which the biomolecule is a thirdsingle-stranded nucleic acid that specifically binds to the firstsingle-stranded nucleic acid in a region other than that which is boundby the second single-stranded nucleic acid; and (c) detectingluminescence, wherein the detection of luminescence indicates that thethird single-stranded nucleic acid of the conjugate bound to the firstsingle-stranded nucleic acid in the sample.

[0042] Preferably, the second single-stranded nucleic acid is anoligonucleotide capture probe, such as a synthetic thymine (poly-T) oradenosine (poly-A) oligonucleotide. Preferably, the secondsingle-stranded nucleic acid is attached to the solid support bystandard crosslinking procedures in accordance with methods known in theart (see, e.g., Joos et al., Anal. Biochem., 247, 96-101 (1997); Runninget al., BioTechniques, 8, 276-277 (1990)). The second single-strandednucleic acid should be of sufficient length and density on the solidsupport so as to bind stably and efficiently with the firstsingle-stranded nucleic acid. Preferably, the capture probe is at leastabout 35 bases in length.

[0043] More broadly, the present invention provides a method ofdetecting a nucleic acid in a sample. The method comprises attaching anucleic acid capture probe to a solid support. The nucleic acid captureprobe comprises a sequence that binds to the nucleic acid in the sample.The attached nucleic acid capture probe is then contacted with thesample, thereby immobilizing the nucleic acid on the solid support. Themethod further comprises contacting the immobilized nucleic acid with aconjugate comprising a water-soluble luminescent semiconductor quantumdot and a biomolecule. The biomolecule of the conjugate specificallybinds to the nucleic acid. Then, the method comprises detectingluminescence. The detection of luminescence indicates that the conjugatebound to the nucleic acid in the sample.

[0044] The present invention also provides a method whereby two or moredifferent molecules and/or two or more regions on a given molecule canbe simultaneously detected in a sample. The method involves using a setof conjugates as described above, wherein each of the conjugates in theset has a differently sized quantum dot or a quantum dot of differentcomposition attached to a biomolecule that specifically binds to adifferent molecule or a different region on a given molecule in thesample. Preferably, the quantum dots of the conjugates range in sizefrom 1 nm to 10 nm, which sizes allow the emission of luminescence inthe range of blue to red. The quantum dot size that corresponds to aparticular color emission is well-known in the art. Within this sizerange, any size variation of quantum dot can be used as long as thedifferently sized quantum dots can be excited at a single wavelength anddifferences in the luminescence between the differently sized quantumdots can be detected. Desirably, the differently sized quantum dots havea capping layer that has a narrow and symmetric emission peak.Preferably, the differently sized quantum dots have an inorganic cappinglayer that matches the structure of the core. More preferably, thedifferently sized quantum dots have a ZnS or a CdSe capping layer.Similarly, quantum dots of different composition or configuration willvary with respect to particular color emission. Any variation ofcomposition between quantum dots can be used as long as the quantum dotsdiffering in composition can be excited at a single wavelength anddifferences in the luminescence between the quantum dots of differentcomposition can be detected. Detection of the different target moleculesin the sample arises from the emission of multicolored luminescencegenerated by the quantum dots differing in composition or thedifferently sized quantum dots of which the set of conjugates iscomprised. This method also enables different functional domains of asingle protein, for example, to be distinguished.

[0045] Accordingly, the present invention provides a method ofsimultaneously detecting two or more different molecules and/or two ormore regions of a given molecule in a sample. The method comprisescontacting the sample with two or more conjugates of a water-solubleluminescent semiconductor quantum dot and a biomolecule, wherein each ofthe two or more conjugates comprises a quantum dot of a different sizeor composition and a biomolecule that specifically binds to a differentmolecule or a different region of a given molecule in the sample. Themethod further comprises detecting luminescence, wherein the detectionof luminescence of a given color is indicative of a conjugate binding toa molecule in the sample.

[0046] In accordance with the present invention, two or more proteins orfragments thereof can be simultaneously detected in a sample.Alternatively, two or more nucleic acids can be simultaneously detected.In this regard, a sample can comprise a mixture of nucleic acids andproteins (or fragments thereof).

[0047] Preferably, in the method of detecting two or more proteins orfragments thereof, the biomolecule of each of the conjugates is aprotein or a fragment thereof, such as an antibody or an antigenicallyreactive fragment thereof, and the proteins or fragments thereof in thesample are antigens or epitopes thereof that are bound by the antibodyor the antigenically reactive fragment thereof. Alternatively and alsopreferably, the biomolecules of each of the conjugates is an antigen orepitope thereof and the proteins or fragments thereof in the sample areantibodies or antigenically reactive fragments thereof that bind to theantigen or epitope thereof. Also preferably, the biomolecule of each ofthe conjugates is a nucleic acid and the proteins or fragments thereofin the sample are nucleic acid binding proteins, e.g., DNA bindingproteins.

[0048] Also, in accordance with the present invention, two or morenucleic acids can be simultaneously detected in a sample. Any of theabove-described methods for detecting a nucleic acid in a sample can beused with two or more conjugates comprising differently sized quantumdots attached to biomolecules that can bind to nucleic acids.Accordingly, one method of simultaneously detecting two or more nucleicacids in a sample comprises (a) contacting the sample with two or moreconjugates, in which each conjugate comprises a differently sizedquantum dot attached to a biomolecule, preferably a nucleic acid, inparticular a single-stranded nucleic acid, or a protein or fragmentthereof, such as a DNA binding protein, that specifically binds to atarget nucleic acid in the sample; and (b) detecting luminescence,wherein the detection of luminescence of a given color indicates that aconjugate bound to its target nucleic acid in the sample.

[0049] Another method of simultaneously detecting two or more nucleicacids in a sample involves using two or more conjugates, each of whichcomprises a different above-described single-stranded oligonucleotidehaving a stem-and-loop structure, in accordance with the methods forusing such a conjugate as set forth above. Yet another method ofsimultaneously detecting two or more nucleic acids in a sample involvesusing the above-described method, wherein the nucleic acids to bedetected are attached to a solid support of the kind described above, inaccordance with the described methods for attaching a nucleic acid in asample and the described methods for detecting said nucleic acid as setforth above. One embodiment of this method is depicted in FIG. 2.

[0050] In another embodiment of the inventive method of simultaneouslydetecting two or more molecules in a sample, the sample comprises atleast one nucleic acid and at least one protein or fragment thereof. Thesimultaneous detection of a nucleic acid and a protein or fragmentthereof in a sample can be accomplished using the methods describedabove in accordance with the described methods for detecting a proteinor fragment thereof in a sample and the described methods for detectinga nucleic acid in a sample as set forth above.

[0051] The above described conjugates and methods can be adapted for usein numerous other methods and biological systems to effect the detectionof a biomolecule. Such methods include, for example, in situhybridization and the like. The present invention also has broadapplication for the real-time observation of cellular mechanisms inliving cells, e.g. ligand-receptor interaction and moleculartrafficking, due to the increased photostability of the quantum dot.

[0052] The present invention has application in various diagnosticassays, including, but not limited to, the detection of viral infection,cancer, cardiac disease, liver disease, genetic diseases, andimmunological diseases. The present invention can be used in adiagnostic assay to detect certain viruses, such as HIV and Hepatitis,by, for example, (a) removing a sample to be tested from a patient; (b)contacting the sample with a water-soluble luminescent quantum dotbiomolecular conjugate, wherein the biomolecule is an antibody orantigenically reactive fragment thereof that binds to the virus; and (c)detecting the luminescence, wherein the detection of luminescenceindicates that the virus is present in the sample. The patient samplecan be a bodily fluid, such as saliva, tears, blood, serum or urine. Forexample, an antibody to HIV gp120 can be used to detect the presence ofHIV in a sample; alternatively, HIV gp120 can be used to detect thepresence of antibodies to HIV in a sample.

[0053] The present invention also can be used in a diagnostic assay todetermine ultra-low-level viral loads of certain viruses, such as HIVand Hepatitis, by detecting the viral nucleic acid. Determining theviral load of a patient is useful in instances where the number of viralparticles is below the detection limits of current techniques. Forexample, this technique can be particularly useful for trackingultra-low HIV levels in AIDS patients during advanced drug treatment,such as triple drug therapy, in which the viral load of the patient hasbeen greatly reduced. The detection of viral nucleic acid can beaccomplished by, for example, (a) removing a sample to be tested from apatient; (b) treating the sample to release the viral DNA or RNA; (c)contacting the sample with a water-soluble luminescent quantum dotbiomolecular conjugate, wherein the biomolecule binds to the nucleicacid of the virus; and (d) detecting the luminescence, wherein thedetection of luminescence indicates that the virus is present in thesample.

[0054] One embodiment of the inventive method is shown in FIG. 3. Usingthis method, the detection of viral nucleic acid is accomplished by (a)removing a sample to be tested from a patient; (b) treating the sampleto release the viral DNA or RNA; (c) attaching capture probes to a solidsupport, wherein the capture probes comprise a sequence that binds tothe viral nucleic acid in the sample; (d) contacting the attachedcapture probes with the viral nucleic acid, thereby immobilizing theviral nucleic acid on the solid support; (e) contacting the immobilizedviral nucleic acid with a luminescent quantum dot conjugate, wherein thebiomolecule of the conjugate specifically binds to the viral nucleicacid; and (f) detecting luminescence, wherein the detection ofluminescence indicates that the conjugate bound to the viral nucleicacid in the sample.

[0055] Preferably, the solid support is a glass surface, a transparentpolymer surface, a membrane, or the like, to which the capture probe canbe attached. The capture probe can be any molecule that is capable ofboth attaching to the solid support surface and binding to the targetviral nucleic acid. Preferably, the capture probe is a single-strandedoligonucleotide comprising a first nucleic acid sequence that binds to acomplementary sequence attached to the solid support and a secondnucleic acid sequence that binds to a third nucleic acid sequence in theviral genome. The oligonucleotide comprising the first and secondnucleic acid sequences can have a length of about 20 to 50 bases.Preferably, the oligonucleotide has a length of at least about 30 bases.Desirably, the third nucleic acid sequence in the viral genome is aconserved sequence.

[0056] The luminescent quantum dot conjugate comprises a luminescentquantum dot attached to a biomolecule that specifically binds to thethird sequence of the target viral nucleic acid in a region other thanthat which is bound by the second sequence of capture probe sequence.The biomolecule can be any molecule that can bind to the target viralnucleic acid. Preferably, the biomolecule is an oligonucleotide thatcontains a fourth sequence that is complementary to the third sequencein the target viral genome. Alternatively, the biomolecule can be a DNAbinding protein that binds specifically to the target viral nucleicacid.

[0057] In addition to the detection of a single virus, the presentinvention can be used to detect simultaneously the viral load of varioustypes of viruses or the viral load of various sub-types of a singlevirus by detecting the different species of viral nucleic acid. Onemethod of simultaneously detecting multiple viral nucleic acids in asample comprises (a) contacting the sample with a set of conjugates,wherein each conjugate of the set comprises a differently sized quantumdot attached to a probe biomolecule that specifically binds to a targetviral nucleic acid in the sample; and (b) detecting the multicoloredluminescence, wherein the detection of multicolored luminescenceindicates that each of the differently conjugates bound to its targetviral nucleic acid in the sample. Yet another method of simultaneouslydetecting two or more nucleic acids in a sample involves using theabove-described method, which is also depicted in FIG. 3.

[0058] The present invention can be used in a similar manner to detectcertain disease states, such as, for example, cancer, cardiac disease orliver disease, by (a) removing a sample to be tested from a patient; (b)contacting the sample with a water-soluble luminescent quantum dotbiomolecular conjugate, wherein the biomolecule is an antibody orantigenically reactive fragment thereof that binds to a proteinassociated with a given disease state, wherein the disease is, forexample, cancer, cardiac disease or liver disease; and (c) detecting theluminescence, wherein the detection of luminescence indicates theexistence of a given disease state. In these cases, the sample can be acell or tissue biopsy or a bodily fluid, such as blood, serum or urine.The protein can be a marker or enzyme associated with a given disease,the detection of which indicates the existence of a given disease state.The detection of a disease state can be either quantitative, as in thedetection of an over- or under-production of a protein, or qualitative,as in the detection of a non-wild-type (mutated or truncated) form ofthe protein. In regard to quantitative measurements, preferably theluminescence of the quantum dot conjugate-target protein complex iscompared to a suitable set of standards. A suitable set of standardscomprises, for example, the luminescent quantum dot conjugate of thepresent invention in contact with various, predetermined concentrationsof the target being detected. One of ordinary skill in the art willappreciate that an estimate of, for example, amount of protein in asample, can be determined by comparison of the luminescence of thesample and the luminescence of the appropriate standards.

[0059] The present invention also can be used to detect a disease state,such as a genetic disease or cancer, by (a) removing a sample to betested from a patient; (b) contacting the sample with water-solubleluminescent quantum dot biomolecular conjugate, wherein the biomoleculeis a nucleic acid that specifically hybridizes with a nucleic acid ofinterest; and (c) detecting the luminescence, wherein the detection ofluminescence indicates the existence of a given disease state. In thesecases, the sample can be a derived from a cell, tissue or bodily fluid.The gene of interest can be a marker for a disease-state, such as BRCAI,which may indicate the presence of breast cancer.

[0060] The above-described methods also can be adapted for in vivotesting in an animal. The conjugate should be administered to the animalin a biologically acceptable carrier. The route of administration shouldbe one that achieves contact between the conjugate and the biomolecule,e.g., protein or nucleic acid, to be assayed. The in vivo applicationsare limited only by the means of detecting luminescence. In other words,the site of contact between the conjugate and the biomolecule to beassayed must be accessible by a luminescence detection means. In thisregard, fiber optics can be used. Fiber optics enable light emission anddetection as needed in the context of the present inventive methods.

EXAMPLES

[0061] The present invention is described further in the followingexamples. These examples serve to illustrate further the presentinvention and are not intended to limit the scope of the invention.

Example 1

[0062] This example demonstrates how to obtain a water-solubleluminescent quantum dot by attaching an attachment group, one end ofwhich can bind to the cap of a luminescent quantum dot and the other endof which comprises a hydrophilic moiety.

[0063] Quantum dots comprising a CdSe core of 4.2 nm and a ZnS cap wereprepared in accordance with the procedure developed by Hines andGuyot-Sionnest (J. Phys. Chem., 100, 468-71 (1996)). The quantum dotswere dissolved in chloroform and reacted with 1.0 M glacialmercapto-acetic acid for 2 hrs at room temperature with slow stirring.Subsequently, an equal volume of aqueous PBS, pH 7.4, was added to thereaction mixture with vigorous shaking for 30 mins. Upon spontaneousseparation of the chloroform and aqueous layers, the aqueous layercontaining the mercaptocoated quantum dots was extracted. The aqueouslayer was then centrifuged to pellet the mercapto-coated quantum dotsand extracted at least four times to remove excess mercapto-acetic acid.The final pellet was resuspended in 10 ml of PBS, pH 7.4, and stored atroom temperature until use. The mercapto-coated quantum dots remainedsoluble for at least one month.

Comparative Example 1a

[0064] This example demonstrates the preparation of a quantum dot whichis not water-soluble.

[0065] Quantum dots having mercaptobenzoic acid groups on the surfacethereof were constructed. Mercaptobenzoic acid was dissolved in asolution of 50% DMSO/50% methanol. The pH of the solution was adjustedto 11 by addition of tetramethyl ammonium hydroxide. The finalconcentration of mercaptobenzoic acid was 5 mM.

[0066] A 1 ml aliquot of the mercaptobenzoic acid solution was added toapproximately 1 mg of the ZnS-capped quantum dots prepared as describedin Example 1 and heated at 70° C. Although the mercaptobenzoicacid-bound quantum dots had temporarily solubilized, the mercaptobenzoicquantum dots began to aggregate rapidly after approximately one hour.Aggregation of quantum dots out of solution is an indication ofinstability. The mercaptobenzoic quantum dots were then purified usingacetone and subsequently placed in PBS. Again, the quantum dots wereunstable and precipitated out of solution after a few hours.

Example 2

[0067] This example demonstrates how to attach a proteinaceousbiomolecule, such as a ligand, to an attachment group on the cap of aluminescent quantum dot by means of a crosslinking agent.

[0068] A 1 ml solution of the purified mercapto-quantum dots of Example1 was reacted with 2 mg of transferrin (Sigma Chemical Co., St. Louis,Mo.) and 1 mg of the crosslinking reagent EDAC (Sigma Chemical Co.)overnight at room temperature while vortexing. The solution was thencentrifuged at 50,000 RPM for 1 hr to pellet the quantum dot-transferrinbioconjugate and the supematant was removed. This centrifugation stepwas repeated twice more. The purified transferrin bioconjugates weredissolved in PBS (pH 7.4) and stored at room temperature.

Example 3

[0069] This example demonstrates how to attach a proteinaceousbiomolecule, such as an antibody, to the attachment group on the cap ofa luminescent quantum dot by means of a crosslinking agent.

[0070] A 1 ml solution of the purified mercapto-quantum dots of Example1 was reacted with 2 mg of Immunoglobulin G (IgG) (Sigma Chemical Co.)and 1 mg of EDAC overnight at room temperature while vortexing. Thesolution was then centrifuged at 50,000 RPM for 1 hr to pellet thequantum dot-immunoglobulin bioconjugate and the supernatant was removed.This centrifugation step was repeated twice more. The purifiedimmunoglobulin bioconjugates were dissolved in PBS (pH 7.4) and storedat room temperature.

Example 4

[0071] This example demonstrates how to attach a nucleic acid to theattachment group on the cap of a luminescent quantum dot by means of alinker, such as a free amine group.

[0072] A 1 ml solution of the purified mercapto-quantum dots of Example1 is reacted with 3′- or 5′-amine-modified oligonucleotides (MidlandCertified Reagents, Midland, Tex.) and 1 mg of EDAC overnight at roomtemperature while vortexing. The solution is then centrifuged at 50,000RPM for 1 hr to pellet the quantum dot-oligonucleotide bioconjugates andthe supernatant is removed. This centrifugation step is repeated twicemore. The purified oligonucleotide bioconjugates are dissolved in PBS(pH 7.4) and stored at room temperature. Using this approach, a directlinkage is formed between the carboxylic acid group on the quantum dotand an amine group on the nucleic acid.

Example 5

[0073] This example demonstrates how to attach a nucleic acidbiomolecule to an attachment group on the cap of a luminescent quantumdot by means of a linker, such as streptavidin or neutravidin.

[0074] Streptavidin (Sigrna Chemical Co.) is covalently linked to themercapto-quantum dots according to the procedures given in Examples 2and 3. After coating the quantum dots with streptavidin, biotinylatedoligonucleotides (Midland Certified Reagents) are incubated with thestreptavidin-coated quantum dots overnight at room temperature withvortexing. The quantum dot-streptavidin-biotinylated-oligonucleotidesare purified by centrifugation at 50,000 RPM for 1 hr. The supernatantis discarded and the pellet is redissolved in PBS (pH 7.4). Thecentrifugation step is repeated twice more. The purified quantumdot-streptavidin-biotinylated-oligonucleotide is dissolved and stored inPBS. The same method can be used substituting neutravidin forstreptavidin.

Example 6

[0075] This example demonstrates the method of using an antibodybioconjugate to detect an antibody in vitro, wherein the method is animmuno-agglutination assay.

[0076] A purified immunoglobulin bioconjugate comprising a water-solubleluminescent quantum dot attached to an immunoglobulin molecule, such asIgG, was prepared by the procedure given in Example 3. The luminescentquantum dot-immunoglobulin bioconjugate was reacted with 0.5 μg/ml ofanti-Fab antibody, which binds to IgG molecules. The reaction mixturewas allowed to incubate for one hour at room temperature. The anti-Fabantibody bound to the IgG molecules of the luminescent quantumdot-immunoglobulin bioconjugate, causing the luminescent bioconjugatesto agglutinate. Agglutination was determined by detecting theluminescence with an epi-fluorescence microscope equipped with ahigh-resolution CCD camera (1.4 million pixels, Photometrix, Tuscon,Ariz.) and a 100 W mercury excitation lamp.

Example 7

[0077] This example demonstrates a method of using a proteinbioconjugate to detect an antibody in vitro, wherein the method is animmuno-agglutination assay.

[0078] A purified protein bioconjugate comprising a water-solubleluminescent quantum dot and a proteinaceous biomolecule, such as anantigen, is prepared according to the procedure in Examples 2 and 3. Apurified cell lysate is prepared from a blood sample by lysing thecells, centrifuging the sample to pellet the cellular debris, and thenextracting the supernatant, which contains the purified cell lysate. Thepurified cell lysate is incubated with the luminescent quantumdot-antigen bioconjugate. If the antibody of interest is present in thecell lysate sample, it will recognize and bind to the antigen attachedto the luminescent quantum dot-antigen conjugate, causing theluminescent quantum dots to agglutinate. Therefore, agglutination of theluminescent quantum dot conjugates indicates the presence of theantibody in the cell lysate sample. Agglutination is determined byluminescence according to the procedure given in Example 6. If desired,duplicate methods can be performed in order to compare the sample to acontrol. A suitable control includes the addition of the luminescentquantum dot-bioconjugate to a physiologically equivalent composition notcomprising the target antibody.

Example 8

[0079] This example demonstrates a method of using an antibodybioconjugate, to detect a protein in vitro, wherein the method is animmuno-agglutination assay.

[0080] A purified antibody conjugate comprising a water-solubleluminescent quantum dot and an antibody is prepared according to theprocedure in Example 3. A purified cell lysate is prepared from a bloodor tissue sample according to the procedure given in Example 7. Thepurified cell lysate is incubated with the luminescent quantumdot-antibody conjugate. If the protein of interest is present in thecell lysate sample, the antibody molecule attached to the luminescentquantum dot-antibody conjugate will recognize and bind to the protein inthe sample, causing the luminescent quantum dots to agglutinate.Agglutination, therefore, indicates the presence of the protein in thesample. The degree of agglutination will indicate the concentration ofprotein present in the cell lysate sample. The degree of agglutinationis determined by luminescence according to the procedure given inExample 6. Of course, to estimate the concentration of protein in asample, the luminescence of the quantum dot-conjugate-protein complex iscompared to a series of standards comprising the luminescent quantumdot-bioconjugate of the present invention in contact with predeterminedconcentrations of target protein.

Example 9

[0081] This example demonstrates a method of using a proteinbioconjugate to detect an antibody in vitro, wherein the method is adirect immunoassay.

[0082] A purified cell lysate is prepared from a blood sample accordingto the procedure given in Example 7. A purified protein bioconjugatecomprising the water-soluble luminescent quantum dot and a proteinaceousbiomolecule, such as an antigen, is prepared according to the procedurein Example 2. The chosen attached antigen is one which is specificallyrecognized by the antibody of interest. A sample of the purified celllysate is pipetted onto a polystyrene surface and allowed to incubatefor two hours at room temperature. The sample is removed and thepolystyrene surface is washed with distilled water. To preventnon-specific binding, a 1% solution of Bovine Serum Albumin (BSA) (SigmaChemical Co.) in PBS is pipetted onto the polystyrene surface andallowed to incubate for one hour at room temperature. After removing theBSA and washing the polystyrene surface with distilled water, thewater-soluble luminescent quantum dot-antigen bioconjugate is pipettedonto the polystyrene surface and allowed to incubate. If the antibody ofinterest is present in the cell-lysate sample, it will recognize andbind to the antigen attached to the luminescent quantum dot-antigenbioconjugate. Luminescence of the quantum dot-antigen bioconjugate isdetected by exciting the sample with an Ar⁺/Kr⁺ laser at 514 nm.

Example 10

[0083] This example demonstrates a method of using an antibodybioconjugate to detect a protein in vitro, wherein the method is asandwich immunoassay.

[0084] A purified cell lysate is prepared from a blood or tissue sampleaccording to the procedure given in Example 7. An antibody bioconjugatecomprising the watersoluble luminescent quantum dot and an antibody isprepared according to the procedure given in Example 3. The chosenattached antibody is one which specifically recognizes the protein ofinterest. First, a “capturing” antibody which recognizes the protein ofinterest is pipetted onto a polystyrene surface and allowed to incubatefor two hours at room temperature. The antibody is removed and thepolystyrene surface is washed with distilled water. To preventnon-specific antibody binding, a 1% solution of BSA in PBS is pipettedonto the polystyrene surface and allowed to incubate for one hour atroom temperature. After removing the BSA and washing the polystyrenesurface with distilled water, the purified cell lysate is pipetted ontothe polystyrene surface and allowed to incubate for two hours at roomtemperature. If the protein of interest is present in the cell-lysatesample, the “capturing” antibody will bind the protein. The polystyrenesurface is washed with distilled water to remove unbound protein.Finally, the luminescent quantum dot-antibody bioconjugate is added andallowed to incubate for two hours at room temperature. If the protein ofinterest is present, the antibody attached to the luminescent quantumdot-antibody bioconjugate will bind to it. Luminescence of the quantumdot-antibody bioconjugate is detected by exciting the sample with anAr⁺/Kr⁺ laser at 514 nm.

Example 11

[0085] This example demonstrates a method of using a nucleic acidbioconjugate to detect a nucleic acid in vitro.

[0086] A purified mRNA sample is prepared from cells or tissue accordingto methods well-known in the art. The particular mRNA of interest willdictate which method and which cells or tissue will be used to isolatethe mRNA. A nucleic acid bioconjugate comprising a water-solubleluminescent quantum dot and a nucleic acid is prepared according to theprocedure given in Examples 4, 5, or 14. The attached nucleic acid iscomprised of an oligonucleotide sequence which specifically hybridizesto the nucleic acid sequence of interest. An amine-modifiedpolythymidine (Midland Certified Reagents) is covalently attached to aBiodyne C membrane (Pall Gelman Sciences, Ann Arbor, Mich.) by adding100 μl of 0.1 μM amine-modified poly-thymidine, one strip of the BiodyneC membrane (0.5 cm×0.5 cm), and 1 mg of EDAC into a centrifuge tube andallowing the mixture to incubate overnight at room temperature. The nextday, the Biodyne C membranes are rinsed with distilled water 3-4 times.The purified mRNA sample is added to the Biodyne C membrane to which theamine-modified poly-thymidine is attached and allowed to incubate fortwo hours at room temperature. The polyA tail of the mRNA molecules willhybridize with the attached poly-thymidine, allowing the mRNA to beattached to the Biodyne C membrane. The Biodyne C membrane is washedseveral times with distilled water to remove non-hybridized mRNA. Next,the oligonucleotide bioconjugate, comprising a water-soluble luminescentquantum dot and an oligonucleotide which specifically hybridizes to themRNA of interest, is reacted with the Biodyne C membrane to which theamine-modified poly-thymidine and mRNA is attached and allowed toincubate overnight at room temperature. If the mRNA of interest ispresent, the luminescent quantum dot-oligonucleotide bioconjugate willhybridize to it. The Biodyne C membrane is washed several times withdistilled water to remove excess non-hybridized luminescent quantumdot-oligonucleotide bioconjugates. Luminescence of the quantumdot-oligonucleotide bioconjugate is detected by exciting the sample withan Ar⁺/Kr⁺ laser at 514 mn.

Example 12

[0087] This example demonstrates a method of using a proteinbioconjugate to detect receptor-mediated endocytosis in vivo.

[0088] A biomolecular conjugate comprising the water-soluble luminescentquantum dot and transferrin was prepared according to the proceduregiven in Example 2. HeLa cells were grown in minimum essential medium(MEM) containing 10% fetal calf serum, 1% penicillin/streptomycin, andfingizone. The cultured cells were incubated with the luminescentquantum dot-transferrin bioconjugates at 37° C. overnight. Afterrepeated washing to remove excess bioconjugate, the cells were removedfrom the petri dish by trypsinization and placed on a glass coverslipfor imaging with an epi-fluorescence microscope equipped with ahigh-resolution CCD camera (1.4 million pixels, Photometrix) and a 100 Wmercury excitation lamp as described in Example 6. Luminescence insideof the HeLa cells indicated that the transferrin of the conjugate wasstill biologically active and was recognized by transferrin receptors onthe HeLa cell surfaces. HeLa cells incubated with the water-solubleluminescent quantum dots of Example 1 were not luminescent. Measurementof the amount of internal luminescence over time enables thedetermination of the rate of endocytosis.

Example 13

[0089] This example demonstrates how to attach a proteinaceousbiomolecule, such as a ligand, to an attachment group on the cap of aluminescent quantum dot by means of a linker, such as streptavidin orneutravidin.

[0090] Streptavidin (Sigma Chemical Co.) is covalently linked to themercaptoquantum dots according to the procedures given in Example 2. Aprotein, such as transferrin, is attached to biotin using the EDACcross-linking method. The attachment to biotin must occur at an aminoacid which can be derivatized with little change in protein activity. Asolution of purified streptavidin-coated quantum dots is reacted withthe biotinylated transferrin overnight at room temperature whilevortexing. The biotinylated transferrin and streptavidin-coated quantumdots can be reacted in a specific molar ratio, such as 1:1, 1:2, etc.,so as produce the desired number of protein molecules per quantum dot.The solution is then centrifuged at 50,000 RPM for 1 hr to pellet thequantum dot-transferrin bioconjugate and the supernatant is removed.This centrifugation step is repeated twice more. The purifiedtransferrin bioconjugates are dissolved in PBS (pH 7.4) and stored atroom temperature.

Example 14

[0091] This example demonstrates how to attach a nucleic acid to theattachment group on the cap of a luminescent quantum dot by means of alinker, such as a thiol group.

[0092] Quantum dots comprising a CdSe core of 4.2 nm and a ZnS cap areprepared in accordance with the procedure developed by Hines andGuyot-Sionnest (1996), supra. Thiol-modified oligonucleotides arepurchased or prepared using standard synthesis procedures. A 1 mlsolution of CdSe(ZnS) quantum dots is reacted with thiol-modifiedoligonucleotides. The ZnS coat of the quantum dot contains unreacted Znmolecules to which the thiol group of the modified oligonucleotide canbind. The solution is then centrifuged at 50,000 RPM for 1 hr to pelletthe quantum dot-oligonucleotide bioconjugates and the supernatant isremoved. This centrifugation step is repeated twice more. The purifiedoligonucleotide bioconjugates are dissolved in PBS (pH 7.4) and storedat room temperature.

Example 15

[0093] This example demonstrates a method of making a specially designednucleic acid bioconjugate which further contains a quencher to detect anucleic acid in vitro.

[0094] The specially designed nucleic acid bioconjugate comprises asingle-stranded oligonucleotide having a stem-and-loop structure, aquantum dot moiety, and a quenching moiety. The oligonucleotide ismodified to have a primary amine group at the 3′ end, whichamine-modified oligonucleotide is available from Midland CertifiedReagents. Using standard cross-linking procedures, the oligonucleotideis further modified to have a biotin group at the 5′ end. Anonfluorescent organic chromophore, 4-[4′-dimethylaminophenylazo]benzoicacid (DABCYL), is covalently linked to the 3′ amino group by using anamino-reactive derivative DABCYL (available from Molecular Probes,Eugene, Oreg.). Quantum dots are first derivatized with strepavidinaccording to the methods described in Example 2 and then conjugated tothe 5′ biotin group at a 1:1 molar ratio. The oligonucleotidebioconjugate is purified using gel-filtration columns and HPLC

Example 16

[0095] This example demonstrates how to obtain a water-solubleluminescent quantum dot by attaching a mercaptosuccinic attachment groupto the cap of a luminescent quantum dot. This example furtherdemonstrates how to attach a nucleic acid to the mercaptosuccinicattachment group by means of a linker, such as a free thiol group.

[0096] Quantum dots comprising a CdSe core of 4.2 nm and a ZnS cap wereprepared as described previously. The quantum dots were dissolved inmercaptosuccinic acid (0.5 μg/ml, pH 9.0) and allowed to mix for 15-30mins at room temperature. A series of acetone precipitations at aconcentration of 30% acetone/70% quantum dot were preformed to purifysufficiently the hydrophilic luminescent quantum dot. The resultingmercaptosuccinic acid-quantum dots were suspended in 15 mM EDAC solutionat pH 6.0.

[0097] A 1 ml solution of the purified mercaptosuccinic-coatedluminescent quantum dots was reacted with either 3′- or5′-thiol-terminated, 15-mer oligonucleotides (Midland CertifiedReagents) at a relative concentration of approximately 100oligonucleotides per quantum dot. The reaction was allowed to proceedfor 30-60 mins while mixing. The luminescent quantum dot-DNA conjugateswere purified several times by acetone precipitation (30% acetone/70%conjugate). The luminescent quantum dot-DNA conjugates were subsequentlystored in a hybridization buffer (0.4 M NaCl, pH 7.0).

Example 17

[0098] This example demonstrates a method of using a nucleic acidbioconjugate to detect a nucleic acid in vitro.

[0099] Equal aliquots of the 3′- and 5′-thiol-terminatedoligonucleotide-luminescent quantum dot conjugates produced as describedin Example 16 were suspended in a hybridization buffer (0.4 NaCl, pH7.0). A complementary 30-mer linker was added to the hybridizationbuffer at a relative concentration of about one linker for every twoluminescent quantum dot conjugates. The 30-mer linker was specificallydesigned to hybridize with the thiol-terminated 15-mer oligonucleotidesof the luminescent quantum dot conjugates. Therefore, if theoligonucleotides of the luminescent quantum dot conjugates retainedtheir biological activity, two quantum dots would hybridize to thelinker oligonucleotide and subsequently aggregate as demonstrated inFIG. 4. Hybridization was monitored for 1-12 hours and aggregation ofthe bioconjugates was observed. Aggregation was imaged using an invertedwide-field Hg lamp excitation and sensitive CCD detection.

[0100] All of the references cited herein, including patents, patentapplications and publications, are hereby incorporated in theirentireties by reference.

[0101] While this invention has been described with an emphasis uponpreferred embodiments, it will be apparent to those of ordinary skill inthe art that variations in the preferred embodiments can be prepared andused and that the invention can be practiced otherwise than asspecifically described herein. The present invention is intended toinclude such variations and alternative practices. Accordingly, thisinvention includes all modifications encompassed within the spirit andscope of the invention as defined by the following claims.

What is claimed is:
 1. A water-soluble luminescent semiconductor quantumdot, which comprises a core, a cap and a hydrophilic attachment group.2. The water-soluble luminescent semiconductor quantum dot of claim 1,wherein the hydrophilic attachment group is attached to said quantum dotvia a sulfur atom.
 3. The water-soluble luminescent semiconductorquantum dot of claim 2, wherein said hydrophilic attachment group is anorganic group comprising a sulfur atom and at least one hydrophilicsubstituent.
 4. The water-soluble luminescent semiconductor quantum dotof claim 3, wherein said hydrophilic substituent is selected from thegroup consisting of a carboxylic acid or salt thereof, a sulfonic acidor salt thereof, a sulfamic acid or salt thereof, an amino substituent,a quaternary ammonium salt, and a hydroxy.
 5. The water-solubleluminescent semiconductor quantum dot of claim 3, wherein said organicgroup is a C₁-C₆ alkyl group or an aryl group.
 6. The water-solubleluminescent semiconductor quantum dot of claim 3, wherein said organicgroup is a C₁-C₆ alkyl group.
 7. The water-soluble luminescentsemiconductor quantum dot of claim 3, wherein said hydrophilicattachment group is a thiol carboxylic acid or thiol alcohol.
 8. Thewater-soluble luminescent semiconductor quantum dot of claim 7, whereinsaid hydrophilic attachment group is mercaptoacetic acid.
 9. Thewater-soluble luminescent semiconductor quantum dot of claim 1, whereinthe core of the quantum dot is selected from the group consisting ofIIB-VIB semiconductors, IIIB-VB semiconductors, and IVB-IVBsemiconductors and the size of the core is from about 1 nm to about 10nm.
 10. The water-soluble luminescent semiconductor quantum dot of claim9, wherein the core of the quantum dot is selected from the groupconsisting of IIB-VIB semiconductors and the size of the core is fromabout 2 nm to about 5 nm.
 11. The water-soluble luminescentsemiconductor quantum dot of claim 10, wherein the core of the quantumdot is CdS or CdSe.
 12. The water-soluble luminescent semiconductorquantum dot of claim 11, wherein the core of the quantum dot is CdSe.13. The water-soluble luminescent semiconductor quantum dot of claim 12,wherein the size of the core is about 4.2 nm.
 14. The water-solubleluminescent semiconductor quantum dot of claim 1, wherein the cap isselected from the group consisting of IIB-VIB semiconductors of highband gap.
 15. The water-soluble luminescent semiconductor quantum dot ofclaim 14, wherein the cap is ZnS.
 16. The water-soluble luminescentsemiconductor quantum dot of claim 11, wherein the cap is ZnS.
 17. Thewater-soluble luminescent semiconductor quantum dot of claim 14, whereinthe cap is CdS.
 18. The water-soluble luminescent quantum dot of claim12, wherein the cap is CdS.
 19. A water-soluble luminescentsemiconductor quantum dot, which comprises a CdSe core, a ZnS cap and amercaptoacetic acid attachment group.
 20. The water-soluble luminescentsemiconductor quantum dot of claim 19, wherein the CdSe core is about4.2 nm and the ZnS coating is about 1 nm.
 21. A composition comprisingthe water-soluble luminescent semiconductor quantum dot of claim 1 andan aqueous carrier.
 22. A composition comprising the water-solubleluminescent semiconductor quantum dot of claim 19 and an aqueouscarrier.
 23. A composition comprising the water-soluble luminescentsemiconductor quantum dot of claim 20 and an aqueous carrier.
 24. Aconjugate comprising the water-soluble luminescent semiconductor quantumdot of claim 1 and a biomolecule, wherein the biomolecule is attached tothe quantum dot via the hydrophilic attachment group.
 25. The conjugateof claim 24, wherein the biomolecule is a protein or a fragment thereof.26. The conjugate of claim 25, wherein the protein or fragment thereofis an antibody or an antigenically reactive fragment thereof.
 27. Theconjugate of claim 24, wherein the biomolecule is a nucleic acid. 28.The conjugate of claim 24, wherein the biomolecule is attached to thehydrophilic attachment group via a linker.
 29. A conjugate comprisingthe water-soluble luminescent semiconductor quantum dot of claim 19 anda biomolecule.
 30. A conjugate comprising the water-soluble luminescentsemiconductor quantum dot of claim 20 and a biomolecule.
 31. Theconjugate of claim 28, wherein the linker is a primary amine.
 32. Theconjugate of claim 28, wherein the linker is streptavidin, neutravidinor biotin.
 33. The conjugate of claim 28, wherein the linker is a thiolgroup.
 34. The conjugate of claim 28, wherein said biomolecule is asingle-stranded oligonucleotide comprising a stem and loop structure andwherein said hydrophilic attachment group is attached to one end of thesingle-stranded oligonucleotide and a quenching moiety is attached tothe other end of the single-stranded oligonucleotide and said quenchingmoiety quenches said luminescent semiconductor quantum dot.
 35. Acomposition comprising the conjugate of claim 24 and an aqueous carrier.36. A composition comprising the conjugate of claim 29 and an aqueouscarrier.
 37. A composition comprising the conjugate of claim 30 and anaqueous carrier.
 38. A method of obtaining a water-soluble luminescentsemiconductor quantum dot, which method comprises: (a) reacting aluminescent semiconductor quantum dot in a nonpolar organic solvent witha first aqueous solution comprising an attachment group; (b) adding asecond aqueous solution of about neutral pH and mixing; and (c)extracting an aqueous layer, thereby obtaining a water-solubleluminescent semiconductor quantum dot.
 39. The method of claim 38,wherein the nonpolar organic solvent is chloroform and the compound ismercaptoacetic acid.
 40. A method of making a conjugate comprising awater-soluble luminescent semiconductor quantum dot of claim 1 and abiomolecule, which method comprises: (a) contacting a water-solubleluminescent semiconductor quantum dot of claim 1 with a biomolecule,which can directly attach to the attachment group on the cap of thewater-soluble luminescent semiconductor quantum dot; and (b) isolatingthe conjugate.
 41. The method of claim 40, wherein said biomolecule is aprotein or a fragment thereof or a nucleic acid.
 42. The method of claim40, wherein said attachment group is mercaptoacetic acid.
 43. The methodof claim 40, wherein (a) further comprises contacting the water-solubleluminescent semiconductor quantum dot and the biomolecule with acrosslinker.
 44. A method of making a conjugate comprising awater-soluble luminescent semiconductor quantum dot of claim 1 and abiomolecule, which method comprises: (a) contacting a water-solubleluminescent semiconductor quantum dot of claim 1 with (i) a linker, anintermediate crosslinker or a bifunctional molecule, and then (ii) abiomolecule, which can indirectly attach to the attachment group on thecap of the water-soluble luminescent semiconductor quantum dot; and (b)isolating the conjugate.
 45. The method of claim 44, wherein saidbiomolecule is a protein or a fragment thereof or a nucleic acid. 46.The method of claim 44, wherein said attachment group is mercaptoaceticacid.
 47. The method of claim 46, wherein said linker is streptavidin,neutravidin or biotin.
 48. A method of making a conjugate comprising awater-soluble luminescent semiconductor quantum dot of claim 1 and abiomolecule, which method comprises: (a) contacting a biomolecule with(i) a linker, an intermediate crosslinker or a bifunctional molecule,and then (ii) a water-soluble luminescent semiconductor quantum dot ofclaim 1; and (b) isolating the conjugate.
 49. The method of claim 48,wherein said biomolecule is a protein or a fragment thereof or a nucleicacid.
 50. The method of claim 49, wherein said attachment group ismercaptoacetic acid.
 51. The method of claim 50, wherein said linker isstreptavidin, neutravidin or biotin.
 52. A method of detecting a proteinin a sample, which method comprises: (a) contacting the sample with aconjugate of claim 24, wherein the biomolecule of the conjugatespecifically binds to the protein; and (b) detecting luminescence,wherein the detection of luminescence indicates that the conjugate boundto the protein in the sample.
 53. The method of claim 52, wherein thebiomolecule of the conjugate is a protein or a fragment thereof.
 54. Themethod of claim 53, wherein the biomolecule of the conjugate is anantibody or an antigenically reactive fragment thereof and the proteinin the sample is an antigen or an epitope thereof that is bound by theantibody or the antigenically reactive fragment thereof.
 55. The methodof claim 54, wherein the antigen or the epitope thereof is viral orbacterial.
 56. The method of claim 53, wherein the biomolecule of theconjugate is an antigen or an epitope thereof and the protein in thesample is an antibody or an antigenically reactive fragment thereof thatbinds to the antigen or epitope thereof.
 57. The method of claim 56,wherein the antibody or the antigenically reactive fragment thereof isspecific for a virus, a bacterium, a part of a virus, or a part of abacterium.
 58. The method of claim 52, wherein the biomolecule of theconjugate is a nucleic acid.
 59. A method of detecting a nucleic acid ina sample, which method comprises: (a) contacting the sample with aconjugate of claim 24, wherein the biomolecule of the conjugatespecifically binds to the nucleic acid; and (b) detecting luminescence,wherein the detection of luminescence indicates that the conjugate boundto the nucleic acid in the sample.
 60. The method of claim 59, whereinthe biomolecule is a nucleic acid.
 61. The method of claim 59, whereinthe biomolecule is a protein or a fragment thereof.
 62. A method ofdetecting a nucleic acid in a sample, which method comprises: (a)contacting the sample with a conjugate of claim 34, wherein said loopcomprises a probe sequence that binds to a target sequence in saidnucleic acid, whereupon the conjugate undergoes a conformational changethat forces the stem to open, thereby separating the quantum dot moietyand the quenching moiety; and (b) detecting luminescence, wherein thedetection of luminescence indicates that the conjugate bound to thenucleic acid in the sample.
 63. A method of detecting a nucleic acid ina sample, which method comprises: (a) contacting a sample comprising afirst single-stranded nucleic acid with a solid support to which isattached a second single-stranded nucleic acid that can bind to saidfirst single-stranded nucleic acid; and (b) contacting said solidsupport with a conjugate of claim 27, in which the biomolecule is athird single-stranded nucleic acid that specifically binds to thefirst-single stranded nucleic acid in a region other than that which isbound by the second single-stranded nucleic acid, and (c) detectingluminescence, wherein the detection of luminescence indicates that thethird single-stranded nucleic acid of the conjugate bound to the firstsingle-stranded nucleic acid in the sample.
 64. A method of detecting anucleic acid in a sample, which method comprises: (a) attaching anucleic acid capture probe to a solid support, wherein said nucleic acidcapture probe comprises a sequence that binds to the nucleic acid in thesample; (b) contacting the attached nucleic acid capture probe with saidsample, thereby immobilizing said nucleic acid on the solid support; (c)contacting the immobilized nucleic acid with a conjugate of claim 27,wherein the biomolecule of the conjugate specifically binds to thenucleic acid; and (d) detecting luminescence, wherein the detection ofluminescence indicates that the conjugate bound to the nucleic acid inthe sample.
 65. A method of simultaneously detecting either two or moredifferent molecules and/or two or more regions of a given molecule in asample, which method comprises: (a) contacting the sample with two ormore conjugates of claim 24, wherein each of the two or more conjugatescomprises a quantum dot of a different size or composition and abiomolecule that specifically binds to a different molecule or adifferent region of a given molecule in said sample; and (b) detectingluminescence, wherein the detection of luminescence of a given color isindicative of a conjugate binding to a molecule in said sample.
 66. Themethod of claim 65, wherein said sample comprises two or more differentproteins or fragments thereof.
 67. The method of claim 65, wherein saidsample comprises two or more different nucleic acids.
 68. The method ofclaim 65, wherein said sample comprises at least one nucleic acid and atleast one protein or fragment thereof.